Substituted β-lactam antibiotics

ABSTRACT

6-Alkoxylated-6-acylamidopenicillanic acids and 7-alkoxylated-7-acylamidocephalosporin acids and esters thereof, are provided by reacting a 6-acylamidopenicillanic acid ester or a 7-acylamidocephalosporin ester under anhydrous conditions at -90° C. to -15° C. with an alkali metal salt of a lower alkyl alcohol in the presence of an excess of the corresponding alcohol to produce, in situ, the anionic form of the antibiotic which on halogenation with a positive halogen compound, e.g. t-butyl hypochlorite, yields the compound of the invention. Compounds of the invention, e.g. 6-methoxy-6-phenoxy-acetamidopenicillanic acid and 7-methoxy-7-[2-(2-thienyl)acetamido]cephalosporanic acid are useful antibiotics.

This is a division of application Ser. No. 545,451 filed Jan. 30, 1975,now U.S. Pat. No. 3,994,885, which was a division of application Ser.No. 301,694 filed Oct. 27, 1972 as a continuation-in-part of applicationSer. No. 222,293 filed Jan. 31, 1972, both now abandoned.

BACKGROUND OF THE INVENTION

This invention relates to the β-lactam antibiotics, the penicillins andthe cephalosporins. In particular, this invention relates to6-alkoxylated 6-acylaminodopenicillanic acids and 7-alkoxylated7-acylamidocephalosporins and to a process for the preparation thereof.

Numerous penicillin and cephalosporin antibiotics have been prepared bysemi-synthetic means. For example, the nucleus of penicillin,6-aminopenicillanic acid (6-APA), has been acylated with a wide varietyof acylating agents to obtain active penicillin antibiotics. Likewise,the cephalosporin nucleus, 7-aminocephalosporanic acid (7-ACA), has beenderivatized in like manner to provide a wide variety of biologicallyactive 7-acylamidocephalosporanic acids. Further, the semi-syntheticnucleus of the cephalosporin antibiotics,7-aminodeacetoxycephalosporanic acid (7-ADCA), has been acylated with awide variety of acyl moieties to provide active7-acylamidodeacetoxycephalosporanic acids. More recently, thecephalosporin nucleus which bears a 3-acetoxymethyl substituent has beenfurther derivatized by the replacement of the acetoxy group with a widevariety of substituents.

Heretofore, cephalosporins and penicillins bearing a substituent on acarbon of the β-lactam ring have been little described in theliterature.

7-Methoxycephalosporin C and7-(5-amino-5-carboxyvaleramido)-7-methoxy-3-carbamoyloxymethyl-3-cephem-4-carboxylicacid obtained from the fermentation of Streptomyces lipmanii andStreptomyces clavuligerus are described in J. Am. Chem. Soc. 93, 2308(1971). 6-Methylpenicillin has been suggested by Strominger, Amer. J.Med., 39, 708 (1965).

It is an object of this invention to provide novel derivatives ofcephalosporin and penicillin antibiotics. It is a further object of thisinvention to provide 6-alkoxy-6-acylamido penicillanic acids and7-alkoxy-7-acylamidocephalosporanic acids and deacetoxycephalosporanicacids. It is also an object of this invention to provide a process forthe preparation of substituted cephalosporins and penicillins whereinthe substituent group is introduced into the 6- and 7-positions of thepenicillin and cephalosporin nuclei, respectively.

SUMMARY OF THE INVENTION

The compounds provided by this invention are represented by thefollowing general formula, ##STR1## wherein the term "acyl" represents awide variety of known side chains of the cephalosporin and penicillinantibiotics such as phenylacetyl, phenylthioacetyl, 2-thiopheneacetyl,phenylglycyl, mandelyl, and the like; R₁ represents hydrogen, an alkalimetal cation, acetoxymethyl or an ester-forming moiety and preferablyone which is readily removed so as to provide the carboxylic acid formof the desired antibiotic; and wherein R₂ represents C₁ -C₄ lower alkyl,allyl, propargyl, C₃ -C₆ cycloalkyl or benzyl; Y is the remaining carbonfragment of the dihydrothiazine ring of the cephalosporin or theremaining carbon fragment of the thiazolidine ring of the penicillin.

As used herein, the term, "cephalosporin," refers to those compoundshaving the 3-cephem ring structure and includes 3-methyl-3-cephemcompounds, the deacetoxycephalosporanic acids, and the3-acetoxymethyl-3-cephem compounds, the cephalosporanic acids. The term,"cephalosporanic acid," has reference to the 3-acetoxymethyl-3-cephemantibiotics.

The compounds of the above general formula are prepared by reacting apenicillanic acid or a cephalosporin acid, an alkali metal salt or anester thereof and preferably an ester which is readily removed toprovide the free carboxylic acid such as the benzyl ester, benzhydrylester or the trichloroethyl ester, in an inert solvent at sub-zerotemperatures and preferably between about -90° C. and -15° C. with analkali metal alkoxide, cycloalkoxide or benzyloxide, in the presence ofan excess of the alcohol corresponding to the alkoxide. To the coldreaction mixture is then added a positive halogen compound such as, forexample, tertiary-butyl hypochlorite or N-chloroacetamide and thereaction mixture stirred in the cold for an additional 5 to 20 minutes.Thereafter the reaction is quenched by the addition of glacial aceticacid or formic acid. The alkoxylated penicillins and cephalosporins canbe recovered from the reaction mixture by conventional isolationprocedures to provide the 6- or 7-substituted penicillin orcephalosporin ester. Removal of the ester group provides a compound ofthe invention represented by the above formula wherein R₁ is hydrogen.

The compounds of the invention exhibit the usual infrared absorptioncharacteristics exhibited by the unsubstituted penicillanic acids andcephalosporanic acids. The compounds of the invention also exhibit thecharacteristic ultraviolet spectra of the penicillanic acids andcephalosporanic acids.

The 6-substituted penicillins and 7-substituted cephalosporins providedby this invention exhibit both gram positive and gram negativemicrobiological activity.

DETAILED DESCRIPTION OF THE INVENTION

The novel substituted penicillins and cephalosporins provided by thisinvention, previously described in general, are represented by thefollowing Formula I. ##STR2## wherein R is hydrogen, C₁ -C₆ alkyl,4-amino-4-carboxybutyl, phenyl, substituted phenyl or a group of theformula ##STR3## wherein P is phenyl, lower alkylphenyl, halophenyl,hydroxyphenyl, lower alkoxyphenyl,

2-thienyl, 3-thienyl, 2-furyl, 3-furyl or 1-tetrazyl;

Z is an oxygen atom, or a sulfur atom;

n is 0 and 1;

a is hydrogen or C₁ -C₃ lower alkyl,

b is hydrogen, C₁ -C₃ lower alkyl, hydroxy, protected hydroxy, amino orprotected amino; and when n is 1, P is phenyl, C₁ -C₄ lower alkylphenyl,halophenyl, hydroxyphenyl, C₁ -C₄ lower alkoxyphenyl and b is hydrogenor C₁ -C₃ lower alkyl;

R₁ is hydrogen, an alkali metal cation, acetoxymethyl or a readilyremovable ester forming moiety;

R₂ is C₁ -C₄ lower alkyl, allyl, propargyl, C₃ -C₆ cycloalkyl or benzyl;

Y is a 3-carbon fragment of the formula ##STR4## or a substituted3-carbon fragment of the formula ##STR5## wherein R₃ is hydrogen, C₂ -C₄alkanoyloxy, C₁ -C₄ alkoxy C₁ -C₄ alkylthio, or pyridinium, ##STR6##wherein R₄, R₅ and R₆ are hydrogen, C₁ -C₄ lower alkyl, phenyl, C₁ -C₄lower alkylphenyl, halophenyl, hydroxyphenyl or C₁ -C₄ loweralkoxyphenyl; or a carbamoyloxy group of the formula ##STR7## wherein R₇and R₈ can be the same or different and are hydrogen or C₁ -C₄ loweralkyl; subject to the limitation that when R₃ is C₂ alkanoyloxy or acarbamoyloxy group wherein R₇ and R₈ are both hydrogen or when Y is the3-carbon fragment ##STR8## then R₂ is C₂ -C₄ alkyl, allyl, propargyl, C₃-C₆ cycloalkyl or benzyl.

The term, "substituted phenyl," has reference herein to phenylsubstituted by one or more substituent groups selected from amongchloro, bromo, fluoro, C₁ -C₄ lower alkyl, for example methyl, ethyl andisopropyl, hydroxy, nitro, amino, aminomethyl, C₁ -C₄ lower alkoxy, forexample, methoxy, ethoxy and isopropoxy, and carboxy. Such substitutedphenyl radicals represented by R can be for example, 4-hydroxyphenyl,3,4-dichlorophenyl, 2,6-dimethoxyphenyl, 4-methylphenyl,4-carboxyphenyl, 3- or 4-nitrophenyl, p-aminophenyl, m-aminophenyl,4-aminomethylphenyl and 4-ethoxyphenyl.

The term, "C₃ -C₆ cycloalkyl," refers to cyclopropyl, cyclobutyl,cyclopentyl and cyclohexyl.

The term, "C₁ -C₄ alkylthio," refers to methylthio, ethylthio,n-propylthio, isopropylthio, n-butylthio and like groups.

The term, "C₁ -C₆ alkyl" as used herein refers to methyl, ethyl,n-propyl, iso-propyl, n-butyl, iso-butyl, t-butyl, n-amyl, iso-amyl,n-hexyl and like aliphatic hydrocarbon radicals. "Lower alkyl" refers tothe C₁ -C₄ straight and branched chain hydrocarbon radicals such asmethyl, ethyl, n-propyl, iso-propyl, n-butyl, sec-butyl, and t-butyl.Likewise, the term, "C₁ -C₃ lower alkyl" has reference to the aliphatichydrocarbon radicals containing from 1 to 3 carbon atoms. The term,"halophenyl" has reference to mono and dichlorophenyl, mono- anddibromophenyl or mono- and difluorophenyl. "Lower alkyl phenyl" refersto the mono- and dialkyl substituted phenyl groups such as methylphenyl,ethylphenyl, n-propylphenyl, iso-propylphenyl, dimethylphenyl,methylethylphenyl and the like. The term, "hydroxyphenyl", refers to themono- or dihydroxy-substituted phenyl group such as p-hydroxyphenyl,m-hydroxyphenyl, 3,4-dihydroxyphenyl and the like. "Lower alkoxyphenyl"refers to the mono- and di-substituted alkyl phenyl ethers such asmethoxyphenyl, ethoxyphenyl, n-propoxyphenyl, 2,6-dimethoxyphenyl andthe like.

The term "protected amino" as employed herein refers to the amino groupsubstituted by one of the commonly employed protecting groups, forexample, t-butyloxycarbonyl, 2,2,2-trichloroethoxycarbonyl,benzyloxycarbonyl, and like protecting groups. "Protected hydroxy"refers to an hydroxyl group substituted by any of the commonly employedhydroxyl protecting groups such as benzyl, benzhydryl,t-butyloxycarbonyl (t-BOC), benzyloxycarbonyl,2,2,2-trichloroethoxycarbonyl, and ethylvinyl ether. It is preferable toemploy an hydroxyl protecting group, such as one of those mentionedabove, which is substantially stable under the basic conditions used inthe present process for the preparation of the penicillin andcephalosporin anionic forms. Any protecting group which is stable underthe conditions of temperature and pH of the process can be used, and theparticular protecting group chosen is not critical in the presentprocess.

The term, "a readily removable ester forming moiety," has reference tothe commonly employed carboxylic acid protecting groups employed forprotecting the C₄ carboxylic acid group of the cephalosporins and the C₃carboxylic acid group of the penicillins. Representative of such groupsare t-butyl, benzyl, benzhydryl, p-nitrobenzyl, 4-methoxybenzyl,2,2,2-trichloroethyl, phenacyl, 3,5-dimethoxybenzyl or tetrahydropyranyland like cleavable ester moieties.

Illustrative of the 7-acyl groups represented by the structural moiety,##STR9## of the formula 1 are formyl, acetyl, propionyl, benzoyl,2,6-dimethoxybenzoyl, 4-hydroxybenzoyl, 3-bromobenzoyl, 4-nitrobenzoyl,4-aminobenzoyl, 3-aminobenzoyl, phenylacetyl, 4-hydroxyphenylacetyl,4-methoxyphenylacetyl, 4-methyl-phenylacetyl, phenoxyacetyl,4-hydroxyphenylacetyl, 3,4-dichlorophenoxyacetyl, 3-methylphenoxyacetyl,phenyl-thioacetyl, 4-chlorophenylthioacetyl, 4-methylphenylthioacetyl,3-bromophenylthioacetyl, mandeloyl, 4-hydroxymandeloyl, phenylglycyl,4-methylphenylglycyl, 4-hydroxy-phenylglycyl, 3-methoxyphenylglycyl,2-thienylacetyl, 3-thienylacetyl, 2-furylacetyl,5-methyl-1-tetrazylacetyl and the like.

Illustrative of the radicals represented by R₃ are methoxy, ethoxy,methylthio, n-propylthio, acetoxy, propionoxy, pyridinium,1-methyl-1H-tetrazol-5-ylthio, 1-phenyl-1H-tetrazol-5-ylthio,1-p-chlorophenyl-1H-tetrazol-5-ylthio;5-methyl-1,3,4-thiadiazol-2-ylthio; 5-phenyl-1,3,4-thiadiazole-2-ylthio;5-methyl-1,3,4-oxadiazol-2-ylthio, 5-phenyl-1,3,4-oxadiazol-2-ylthio,carbamoyloxy, N-methylcarbamoyloxy, N,N-dimethylcarbamoyloxy,N,N-diethylcarbamoyloxy, N-butylcarbamoyloxy andN-methyl-N-ethylcarbamoyloxy.

The following compounds are illustrative of the 7-substituted7-acylamido cephalosporin compounds provided by the present invention.

7-acetamido-7-methoxy-3-methyl-3-cephem-4-carboxylic acid,

7-methoxy-3-methyl-7-(2-phenylacetamido)-3-cephem-4-carboxylic acid,

7-ethoxy-7-(2-phenoxyacetamido)cephalosporanic acid,

7-formamido-7-methoxy-3-methyl-3-cephem-4-carboxylic acid,

7-allyloxy-3-methyl-7-(2-phenylacetamido-3-cephem-4-carboxylic acid,

7-isopropoxy-7-[2(2-thienyl)acetamido]cephalosporanic acid,

7-methoxy-3-methyl-7-[2-(2-thienyl)acetamido]-3-cephem-4-carboxylicacid,

7-n-butoxy-7-(2-phenylacetamido)cephalosporanic acid,

7-methoxy-3-methyl-7-(2-phenylthioacetamido)-3-cephem-4-carboxylic acid,

7-[2-(m-hydroxyphenyl)acetamido]7-isopropoxy-3-methyl-3-cephem-4-carboxylicacid,

7-(5-amino-5-carboxyvaleramido)-7-ethoxycephalosporanic acid,

7-(5-amino-5-carboxyvaleramido)-7-benzyloxycephalosporanic acid,

7-ethoxy-3-methyl-7-[2-(3-thienyl)acetamido]-3-cephem-4-carboxylic acid,

7-[2-(2-furyl)acetamido]-7-isopropoxycephalosporanic acid,

7-n-butoxy-7-[2-(4-chlorophenyl)acetamido]-3-methyl-3-cephem-4-carboxylicacid,

7-methoxy-3-methoxymethyl-7-(2-phenylacetamido)-3-cephem-4-carboxylicacid,

7-methoxy-3-methylthiomethyl-7-(2-phenoxyacetamido)-3-cephem-4-carboxylicacid,

3-carbamoyloxymethyl-7-n-propoxy-7-[2-(2-thienyl)acetamido]-cephem4-carboxylic acid,

7-n-butoxy-7-n-butyramido-3-methoxymethyl-3-cephem-4-carboxylic acid,

7-benzamido-7-methoxy-3-(N-methylcarbamoyloxymethyl)-3-cephem-4-carboxylicacid,

7-(2-amino-2-phenylacetamido)-7-methoxy-3-methyl-3-cephem-4-carboxylicacid,

7-(2-t-butoxycarbonylamino-2-phenylacetamido)-7-ethoxy-3-methyl-3-cephem-4-carboxylicacid;

7-mandelamido-7-methoxy-3-methyl-3-cephem-4-carboxylic acid,

7-(2-benzhydryloxy-2-phenylacetamido-7-methoxy-3-methyl-3-cephem-4-carboxylicacid,

7[2-amino-2-(p-hydroxyphenyl)acetamido]-7-methoxy-3-methyl-3-cephem-4-carboxylicacid,

7-cyclopropoxy-3-methyl-7-(2-phenylacetamido)-3-cephem-4-carboxylicacid,

7-cyclopropoxy-3-methyl-7-(2-phenoxyacetamido)-3-cephem-4-carboxylicacid,

7-(2-amino-2-phenylacetamido)-7-cyclopentoxy-3-ethoxymethyl-3-cephem-4-carboxylicacid,

7-(2-phenylacetamido)-7-propargloxycephalosporanic acid,

7-isopropoxy-3-propionyloxymethyl-7-[2-(2-thienyl)acetamido]3-cephem-4-carboxylic acid,

7-[2-amino-2-(3-thienyl)acetamido]-7-methoxy-3-methyl-3-cephem-4-carboxylicacid,

7-[2-(2,6-dimethoxyphenyl)acetamido]-7-methoxy-3-methyl-3-cephem-4-carboxylicacid,

7-[2-(p-bromophenyl)acetamido-3-carbamoyloxymethyl-7-isopropoxy-3-cephem-4-carboxylicacid,

7-[2-(3,4-dimethoxyphenyl)acetamido]-3-isopropoxymethyl-7-ethoxy-3-cephem-4-carboxylicacid,

7-(5-amino-5-carboxyvaleramido)-7-methoxy-3-methyl-3-cephem-4-carboxylicacid,

7-methoxy-3-(5-methyl-1,3,4-oxadiazol-2-ylthiomethyl)-7-[2-(2-thienyl)acetamido]-3-cephem-4-carboxylicacid,

7-cyclopropoxy-3-(1-methyl-IH-tetrazol-5-ylthiomethyl)-7-(2-phenoxyacetamido)-3-cephem-4-carboxylicacid,

7-ethoxy-3-(5-methyl-1,3,4-thiadiazol-2-ylthiomethyl)-7-(2-hydroxy-2-phenylacetamido)-3-cephem-4-carboxylicacid,

7-allyloxy-3-(1,3,4-thiadiazol-2-ylthiomethyl)-7-(2-phenylacetamido)-3-cephem-4-carboxylicacid,

7-methoxy-7-[2-(1-tetrazyl)acetamido]-3-methoxymethyl-3-cephem-4-carboxylicacid,

7-methoxy-7-[2-(1-tetrazyl)acetamido]-3-methylthiomethyl-3-cephem-4-carboxylicacid,

7-methoxy-3-(5-phenyl-1,3,4-thiadiazol-2-ylthiomethyl)-7-[2-(2-thienyl)acetamido]-3-cephem-4-carboxylicacid,

7-methoxy-3-[1-(4-chlorophenyl)-IH-tetrazol-5-ylthiomethyl]-7-[2-amino-2-(4-hydroxyphenyl)acetamido]-3-cephem-4-carboxylicacid, and the like.

7-methoxy-7-mandelamido-3-(N,N-dimethylcarbamoyloxymethyl)-3-cephem-4-carboxylicacid,

7-methoxy-7-[2-(2-thienyl)acetamido]-3-(N-methyl-carbamoyloxymethyl)-3-cephem-4-carboxylicacid, and the like.

The following 6-substituted 6-acylamidopenicillanic acids arerepresentative of those provided by the present invention:

6-(2,6-dimethoxybenzamido)-6-ethoxypenicillanic acid,

6-(2-phenylacetamido-6-propoxypenicillanic acid,

6-allyloxy-6-butyramidopenicillanic acid,

6-(2-phenylacetamido-6-propargyloxypenicillanic acid,

6-cyclopropoxy-6-(2-phenylacetamido)penicillanic acid,

6-cyclohexyloxy-6-(2-phenoxyacetamido)penicillanic acid,

6-[2-(3,4-dichlorophenyl)acetamido]-6-isopropoxypenicillanic acid,

6-ethoxy-6-[2-(p-ethoxyphenyl)acetamido]penicillanic acid,

6-(2-methyl-2-phenylacetamido)-6-n-propoxypenicillanic acid,

6-benzyloxy-6-[2-(2-thienyl)acetamido penicillanic acid,

6-(2-amino-2-phenylacetamido)-6-methoxypenicillanic acid,

6-cyclopropoxy-6-(2-phenylthioacetamido)penicillanic acid,

6-[2-(p-bromophenoxy)acetamido]-6-n-butoxypenicillanic acid and thelike.

Preferred compounds of the present invention are those represented bythe Formula I wherein R₂ is methyl. An especially preferred group ofcompounds provided by this invention are those compounds represented bythe formula I wherein R₂ is methyl, Y is the substituted 3-carbonfragment ##STR10## wherein R₃ is ##STR11## and R₄ is other thanhydrogen. These especially preferred compounds are represented by theformula. ##STR12## wherein R₄ is other than hydrogen. Illustrativecompounds represented thereby are7-methoxy-7-[2-(1-tetrazyl)acetamido]-3-(1-methyl-1H-tetrazole-5-ylthiomethyl)-3-cephem-4-carboxylicacid,7-methoxy-7-mandelamido-3-(1-methyl-1H-tetrazole-5-ylthiomethyl)-3-cephem-4-carboxylicacid, and

7-methoxy-7-[2-(2-thienyl)acetamido]-3-(1-methyl-1H-tetrazol-5-ylthiomethyl)-3-cephem-4-carboxylicacid, and the acetoxymethyl esters and alkali metal salts thereof.

As is the case with non-alkoxylated penicillanic acids andcephalosporanic acids the compounds of this invention readily form saltssuch as the lithium, sodium, and potassium salts by the reaction of thefree antibiotic acid in a suitable solvent with an alkali metalcarbonate or bicarbonate.

The compounds represented by the Formula I, wherein R₁ is acetoxymethyl,are ester derivatives of the penicillanic and cephalosporin free acidswhich possess the antibiotic activity of the free acid forms of theantibiotics and are relatively stable esters. In contrast, the readilyremovable esters represented when R₁ is a readily removable esterforming moiety, for example the p-nitrobenzyl or 2,2,2-trichloroethylesters, are themselves substantially inactive antibiotic compounds.However, such esters are useful intermediates in the preparation of theantibiotic free acids in the process of the invention as describedhereinafter.

According to another aspect of the present invention, a novel process isprovided for the preparation of the compounds represented by the FormulaI. According to this invention, a 6-acylamidopenicillanic acid ester ora 7-acylamidocephalosporin ester represented by the Formula II ##STR13##wherein R, and Y have the same meanings as defined in Formula I exceptthat when Y is a substituted 3-carbon fragment of the formula ##STR14##R₃ is hydrogen, C₂ -C₄ alkanoyloxy, C₁ -C₄ alkoxy, C₁ -C₄ alkylthio;##STR15## or a carbamoyloxy group of the formula wherein R₄, R₅, R₆, R₇and R₈ have the same meanings as defined in Formula I; and R₉ isacetoxymethyl or a readily removable ester forming group; is reacted inan inert, anhydrous, solvent with an alkali metal salt of a C₁ -C₄ alkylcarbinol, allyl alcohol, propargyl alcohol, a C₃ -C₆ cycloalkylcarbinol, or benzyl alcohol, represented by the formula

    M.sup.+ OR.sub.2.sup.-

wherein R₂ is C₁ -C₄ lower alkyl, C₃ -C₆ cycloalkyl, allyl, propargyl orbenzyl and M⁺ is an alkali metal cation, in the presence of an excessamount of the corresponding alcohol, HOR₂, at a temperature betweenabout -95° C. and about -15° C. to provide the anionic form of thecompound of Formula II. To the reaction solution of the anionic form isadded a halogenating agent which under the conditions of the reaction iscapable of supplying positive halogen. The reaction mixture is allowedto stir at the reaction temperature for about 5 to 15 minutes and isthereafter quenched with a carboxylic acid such as formic acid orglacial acetic acid to provide a reaction product mixture containing the6-substituted penicillanic acid ester or the 7-substituted cephalosporinester. The reaction mixture is evaporated in vacuo to dryness and theresidue is dissolved in a suitable organic solvent. The solution is thenwashed with a weak base such as sodium bicarbonate and then dried andthe dried solution evaporated to yield the crude reaction productmixture containing the desired product. The product is purified byrecrystallization or perferably by chromotagraphy over silica.

Alkali metal salts of the alcohols which can be employed in the presentprocess include for example lithium methoxide, potassium methoxide,sodium methoxide, lithium ethoxide, lithium allyloxide, lithiumcyclopropoxide, lithium propargyloxide, the lithium, sodium andpotassium salts of benzyl alcohol and the like.

In general, the alkali metal salts of the alcohols are prepared in situin the presence of an excess amount of the corresponding alcohol in theinert, dry solvent, and the starting penicillanic acid ester orcephalosporin ester is added thereto to generate the anion. The amountof the alkali metal salt which can be employed in the present process isbetween about 2 and 6 equivalents per 1 equivalent of the penicillin orthe cephalosporin employed. The preferred amount is about 3.5equivalents of alkali metal salt to one equivalent of the antibiotic.

Inert solvents which are suitable in the present process includetetrahydrofuran, dioxane, dimethylformamide, dimethylacetamide, thedimethylether of ethylene glycol, and polyethers such as diethyleneglycol dimethyl ether. The particular solvent employed in the presentprocess is unimportant and any suitable inert solvent can be employed.However, it is a requirement of the present process that the solventemployed to anhydrous since minor amounts of water drastically reducethe yield of the desired product.

As previously mentioned the reaction of an alkali metal salt of analcohol, M⁺ OR₂ ⁻, with the antibiotic ester is carried out in thepresence of an excess of the corresponding alcohol. By an excess amountis meant an amount greater than 1 equivalent of the antibiotic employedand generally an excess between 10 and 30 equivalents can be used sincesuch excess is not critical in the process.

The halogenating agents which can be employed in the present process arethose which are generally recognized as positive halogen sources. Theterm, "positive halogen source," refers to any halogen compound capableof supplying a positive halogen atom, for example, Cl⁺, Br⁺ and I⁺. Awide variety of such compounds are known and recognized as sources ofpositive halogens. For example, chlorine, bromine, N-haloamides andN-haloimides such as N-chlorosuccinimide, N-bromosuccinimide,N-chloroacetamide, the N-halosulfonamides such asN-chloro-benzenesulfonamide and N-chloro-p-toluenesulfonamide, the1-halobenzotriazoles such as 1-bromobenzotriazole, also thehalotriazines, the organic hypochlorites such as t-butyl hypochloriteand t-butyl hypoiodide, the halohydantoins, such as N,N-dibromohydantoincan be employed as positive halogen sources. A preferred halogenatingagent, acting as a source of positive chlorine in the present process,is t-butyl hypochlorite.

The halogenating agent is desirably used in an amount sufficient toprovide 1 equivalent of positive halogen per equivalent of theantibiotic employed in the reaction.

The entire process is carried out over a relatively short period oftime. For example, upon the addition of the cephalosporanic acid esteror the penicillanic acid ester to a solution of the alkali metal salt ofthe alcohol in an inert solvent, the anion is generated rapidly andthereafter the halogenating agent can be added to the reaction mixturealmost immediately. The reaction mixture is maintained in the coldbetween about 5 and 20 minutes and is thereafter quenched byacidification.

Suitable acids which can be employed in the acidification are thosewhich when added to the cold reaction mixture do not cause freezing ofthe reaction mixture into a solid or heavy viscous mixture. Suitableacids are the low molecular weight alkyl carboxylic acids such as formicacid, acetic acid, or propionic acid, the chlorinated alkyl carboxylicacids such as trichlorocetic acid, and the lower alkylsulfonic acidssuch as methanesulfonic acid. Aqueous mineral acids are undesirable dueto the high content of water which rapidly freezes in the cold reactionmixture. Glacial acetic acid and 98% formic acid are the preferred acidsin the present process.

The reaction is usually carried out by first preparing a solution of thealkali metal salt of the desired alcohol in an excess of thecorresponding alcohol in an inert anhydrous, solvent such astetrahydrofuran and cooling the mixture to the reaction temperature.Thereafter, a solution of the desired penicillin or cephalosporincompound in anhydrous tetrahydrofuran is added rapidly by dropwiseaddition to the reaction vessel. The reaction mixture is stirred for ashort time, and generally only about 3 minutes is required to generatethe anionic form, and thereafter the halogenating agent is added rapidlyto the solution of the antibiotic anion. The reaction mixture is thenstirred for an additional 5 or 10 minutes at the reaction temperatureand is thereafter quenched by the addition of an excess amount of formicacid or acetic acid. The reaction can be carried out, however, overlonger periods of time provided the temperature is maintained at thereaction temperature.

Following treatment of the reaction mixture with formic acid or glacialacetic acid, the reaction mixture is evaporated to dryness and theresidue is dissolved in a suitable solvent such as a halogenatedhydrocarbon, for example, dichloromethane, chloroform, or carbontetrachloride, or an ester such as ethyl acetate or amyl acetate orother suitable solvent. This solution is washed with a saturatedsolution of sodium thiosulfate to remove excess halogen, and then with asaturated solution of sodium bicarbonate to remove any residual acidwhich may be present. The washed solution is then dried and thereafteris evaporated to dryness in vacuo to yield the reaction product mixturewhich occasionally contains some unreacted starting material. Thesubstituted antiobiotic and the starting material can be convenientlyseparated from each other by chromatography. Suitable chromatographicmaterials which can be employed in the separation of the alkoxylatedantiobiotic from its starting material are silica gel and alumina.Likewise, when the reaction is carried out on a small scale, thereaction product can be isolated by means of preparative thin layerchromatography.

The foregoing process is conveniently illustrated by the followingreaction scheme. ##STR16## wherein R, R₂, R₉, and Y have the samemeanings as previously defined and M = Li, Na, K, and THF =tetrahydrofuran.

The preferred conditions and reagents of the above-described process areas follows.

It is preferred to employ an ester of a 6-acylamidopenicillanic acid ora 7-acylamidocephalosporin as the starting material in the presentinvention, a particularly useful ester being the 2,2,2-trichloroethylester.

Although the free carboxylic acid form of the starting penicillin orcephalosporin can be used, yields of the reaction products are higherthan when an ester is employed.

Tetrahydrofuran is the preferred solvent, t-butyl hypochlorite is thepreferred halogenating agent and the reaction is carried out preferablyat about -80° C. When the reaction is carried out in the preferredmanner and with the preferred reagents, yields of the substitutedantibiotics on the order of between about 40 and 95 percent are commonlyobtained.

The alkoxylated β-lactam compounds provided by this invention havespectral properties analogous to those of the non-alkoxylatedpenicillins and cephalosporins. For example, the presence of theβ-lactam ring carbonyl is shown in the products by the presence of anabsorption maximum in the infrared at approximately 1790 cm⁻¹. Thecompounds of this invention likewise exhibit the characteristicultraviolet absorption spectra of the unsubstituted penicillins andcephalosporins. The nuclear magnetic resonance spectra of the compoundsof the invention, as expected, show the absence of the peaksattributable to the hydrogens attached to the adjacent carbon atoms ofthe β-lactam ring, for example, in the penicillins, the hydrogensattached to C₅ and C₆, and in the cephalosporins the adjacent hydrogensattached at C₆ and C₇. Instead, the spectra of the compounds of theinvention show only a singlet for the remaining proton attached to thecarbon of the ring juncture resulting from the C₅ proton of thepenicillin and the C₆ proton of the cephalosporin.

When in Formula I, R is 4-amino-4-carboxybutyl, it is preferable toprotect the free amino group of the side chain (aminoadipoyl) during thesubstitution reaction of the present process. Commonly employed aminoprotecting groups can be used in the present process, for example,t-butyloxycarbonyl, benzyloxycarbonyl, 2,2,2-trichloroethoxycarbonyl andlike amino protecting groups can be employed. Likewise, when R in theFormula I is 2-hydroxy-2-phenylacetamido, the mandelic acid side chain,the hydroxy group is protected during the substitution reaction of thepresent invention. The hydroxy group can be conveniently protected bysuch groups as benzyl, benzhydryl, tetrahydropyranyl, ethyl vinyl etherand like hydroxyl protecting groups.

As previously mentioned, the starting material in the present process ispreferably an ester of the desired penicillin or cephalosporin. Whereasany convenient ester group can be used, it is desirable that the estergroup chosen be one which is readily removed following the reactionprocess so as to provide easy access to the reaction products in thefree acid form. Consequently, the particular ester chosen isconveniently one which has been previously employed as a carboxylic acidprotecting group in the penicillin and cephalosporin art. Such estermoieties, which are readily cleavable include the p-nitrobenzyl, benzyl,benzhydryl, p-methoxybenzyl, 2,2,2-trichloroethyl, the esters formedwith phenacyl halides such as phenacyl bromide, the tertiary butyl esterand like esters.

Such esters are removed according to published methods, for example, the2,2,2-trichloroethyl ester is removed with zinc and acetic or formicacids as described in R. B. Woodward, et al., J. Am. Chem. Soc. 88, 852(1966). The p-nitrobenzyl group is removed by hydrogenolysis underacidic conditions. The diphenylmethyl ester (benzhydryl) can be removedwith trifluoroacetic acid in anisole at about 0 - 10° C. as described byBritish Pat. No. 1,041,985. The benzyl ester moiety can be removed bycatalytic hydrogenolysis with hydrogen in the presence of palladium oncarbon catalyst, for example, as described by U.S. Pat. No. 3,197,466.The t-butyl ester is removed according to the method described by J.Org. Chem., 31, 444 (1966). The p-methoxybenzyl ester can be removed byfollowing the procedure described by R. R. Chauvette, et al., J. Org.Chem., 36, 1259 (1971).

The compounds of the invention, where R₃ of the substituted 3-carbonfragment y in the Formula I is a 2-thiotetrazole, a2-thio-1,3,4-thiadiazole or a 2-thio-1,3,4-oxadiazole substituent, areprepared in an alternative method by first reacting a7-acylamidocephalosporanic acid ester (Formula II, R₃ =acetoxy) in theprocess of the invention to obtain a 7-alkoxy substitutedcephalosporanic acid ester intermediate. The 7-alkoxy substitutedintermediate is thereafter reacted according to the conditions andprocedures described in U.S. Patent 3,516,997 to effect the nucleophilicdisplacement of the 3-acetoxy substituent with the thiotetrazole,thio-1,3,4-thiadiazole or thio-1,3,4-oxadiazole. For example,p-nitrobenzyl 7-[2-(2-thienyl)acetamido]cephalosporanate is reacted indry tetrahydrofuran at -80° C. with methyl lithium, excess methanol andt-butyl hypochlorite to provide p-nitrobenzyl7-methoxy-7-[2-(2-thienyl)acetamido]cephalosporanate. The reactionproduct is isolated and is then reacted with hydrogen in an inertsolvent in the presence of 5% Pd/c catalyst to effect hydrogenolysis ofthe p-nitrobenzyl ester and provide7-methoxy-7-[2-(2-thienyl)acetamido]cephalosporanic acid. The latterproduct is then converted to its sodium salt in a dilute solution ofsodium bicarbonate and the sodium salt reacted in a mixture of acetoneand water containing sodium bicarbonate with1-phenyl-IH-tetrazole-5-thiol to obtain7-methoxy-3-(1-phenyl-IH-tetrazol-2-ylthiomethyl)-7-[2-(2-thienyl)acetamido]-3-cephem-4-carboxylicacid sodium salt.

The compounds of the invention, represented by the Formula I, wherein R₃of the substituted 3-carbon fragment Y is pyridine, are prepared byheating a 7-alkoxy (R₂ O-) cephalosporanic acid (R₃ is acetoxy) withpyridine in an inert solvent, for example acetone. Accordingly the7-alkoxy, 7-cycloalkoxy, 7-allyloxy, 7-propargyloxy and7-benzyloxycephalosporanic acids prepared in the present process can beconverted to the corresponding pyridinium antibiotics. For example,7-methoxy-7-[2(2-thienyl)acetamido]cephalosporanic acid is heated inacetone with pyridine to provide the 7-methoxy derivative of the wellknown antibiotic cephaloridine.

The starting materials (Formula II) in the process of this invention areknown compounds or they can be prepared by methods known in the art. Forexample, the cephalosporanic acids, R₃ =acetoxy, are prepared by theacylation of 7-aminocephalosporanic acid (7-ACA) with the desired acylgroup, ##STR17## Compounds wherein R₃ is a C₃ -C₄ alkanoyloxy group canbe prepared by the acylation of a 7-acylamidodesacetylcephalosporanicacid ester with a C₃ - or C₄ -alkanoic acid halide in the presence of ahydrogen halide acceptor. Likewise, the 3-carbamoyloxymethyl substitutedcompounds, ##STR18## are prepared with the desacetylcephalosporins andthe appropriate carbamoyl halide. The starting compounds, wherein R₃ isC₁ -C₄ alkoxy, are prepared by reacting a 3-bromomethylcephalosporinwith the appropriate C₁ -C₄ alkyl metal alkoxide, for example, sodiummethylate. Compounds wherein R₃ is C₁ -C₄ thioalkyl, are prepared byreacting a cephalosporanic acid with a C₁ -C₄ alkyl mercaptan in thepresence of a weak base.

The acetoxymethyl esters of the compounds of Formula II, R₉ =acetoxymethyl, can be prepared with the corresponding cephalosporin acidor penicillanic acid and a halomethyl acetate, such as chloromethylacetate according to the method described in The Journal of Antibiotics,XXIV No. 11, 771 (1971).

Illustrative of the starting materials which can be employed in thepresent process are the acetoxymethyl and readily cleavable esters ofthe following acids: 6-Acetamidopenicillanic acid,6-benzamidopenicillanic acid, 6-(2,6-dimethoxybenzamidopenicillanicacid, 6-phenylacetamidopenicillanic acid (Pen. G),6-phenoxyacetamidopenicillanic acid (Pen V),6-D-phenylglycylamidopenicillanic acid, 7-acetamidocephalosporanic acid,cephalosporin C, 7-acetamido-3-methyl-3-cephem-4-carboxylic acid,7-phenoxyacetamidocephalosporanic acid, 7-mandelamidocephalosporanicacid, 7-D-phenylglycylamidocephalosporanic acid (cephaloglycin),7-D-phenylglycylamido-3-methyl-3-cephem-4-carboxylic acid (cephalexin),7-[2-(2-thienyl)acetamido]cephalosporanic acid (cephalothin),7-mandelamido-3-(1-methyl-1H-tetrazole-5-ylthiomethyl)-3-cephem-4-carboxylicacid,7-[2-(2-thienyl)acetamido]-3-(5-methyl-1,3,4-thiadiazol-2-ylthiomethyl)-3-cephem-4-carboxylicacid, 7-phenoxyacetamido-3-methoxymethyl-3-cephem-4-carboxylic acid,7-phenoxyacetamido-3-methylthiomethyl-3-cephem-4-carboxylic acid,7-[2-(2-thienyl)acetamido-3-N,N-dimethylcarbamoyloxymethyl-3-cephem-4-carboxylicacid, and 7-propionamido-3-(5-methyl-1,3,4-oxadiazol-2-ylthiomethyl)-3-cephem-4-carboxylic acid.

According to a further object of the present invention when a compoundof the Formula II, wherein ##STR19## represents the acyl moiety derivedfrom mandelic acid or a substituted mandelic acid having an unprotectedhydroxyl group, is employed in the process of this invention anintramolecular alkoxylation involving the unprotected hydroxyl groupresults to provide a spiro β-lactam compound of the Formula III.##STR20## wherein R₉ and Y have the same meanings as defined in FormulaII and R₁₀ represents phenyl, halophenyl, lower alkylphenyl, loweralkoxyphenyl an hydroxyphenyl as previously defined. For example,benzhydryl7-(2-hydroxy-2-phenylacetamido)-3-carbamoylmethyl-3-cephem-4-carboxylateis added to methyl lithium and excess methanol in dry tetrahydrofuran ata temperature of -78° C. and 1.2 equivalents of t-butyl hypochlorite areadded to the cold reaction mixture. The mixture is stirred in the coldfor 3 minutes and is then acidified with 98% formic acid to provide thecompound of Formula III wherein R₁₀ is phenyl and R₉ is benzhydryl.Hydrogenolysis of the benzhydryl ester affords the free acid,3-carbamoyloxymethyl-4'-oxo-5'-phenylspiro[3-cephem-7,2'-oxazolidine]4-carboxylic acid of the formula##STR21##

The spiro β-lactam esters of the Formula III are converted to thecorresponding acids by removal of the ester forming group R₉ accordingto conventional procedures such as hydrogenolysis and acid hydrolysis.

The spiro β-lactam acids are useful antibiotic compounds possessingsignificant activity against the gram positive organisms. Whenadministered parenterally the spiro β-lactam acids of the alkali metalsalts thereof, for example the sodium salt, are useful in combattinggram positive infections in warm blooded mammals.

Illustrated of the spiro β-lactam compounds provided by this inventionare the following:

3-acetoxymethyl-4'-oxo-5'-phenylspiro[3-cephem-7,2'-oxazolidine]-4-carboxylicacid,

3-methyl-4'-oxo-phenylspiro-[3-cephem-7,2'-oxazolidine]-4-carboxylicacid,

3-acetoxymethyl-4'-oxo-5'-(4-chlorophenyl)spiro-[3-cephem-7,2'-oxazolidine]4-carboxylicacid,

benzhydryl3-methyl-4'-oxo-5'-(4-hydroxyphenyl)spiro[3-cephem-7,2'-oxazolidine]4-carboxylate,

benzhydryl2,2-dimethyl-4'-oxo-5'-phenylspiro[penam-6,2'oxazolidine]-3-carboxylate,

2,2-dimethyl-4'-oxo-5'-(4-methoxyphenyl)-spiro-[penam-6,2'-oxazolidine]-3-carboxylicacid, and like spiro-oxazolidinone penicillins and cephalosporins.

The preparation, in situ, of an anionic form of the6-acylamidopenicillanic acid esters and the 7-acylamido-3-substitutedmethyl-3-cephem-4-carboxylic acid esters, the cephalosporin esters, is asignificant aspect of the present invention. The preparation of suchanionic forms of these antibiotics followed by the reaction of the anionwith a positive halogen makes possible the subsequent nucleophilicsubstitution on the β-lactam carbon atom.

It will be appreciated by those skilled in the art that other aniongathering bases can be employed in the initial phase of the abovedescribed process for the preparation of the anionic forms of thepenicillins and cephalosporins. One such anion generator which can beemployed is lithium diisopropyl amide.

The compounds provided by the process of this invention are usefulantibiotic substances which inhibit the growth of microorganismspathogenic to animal and plant life. For example, in standard in vitrotests, the compounds of the invention demonstrate significant activityagainst the following illustrative microorganisms: Staphylococcusaureus, Bacillus subtilis, Sarcina lutea, Escherichia coli, Klebsiellapneumoniae, Proteus vulgaris, Salmonella gallinarium, Serratiamarcescens and Pseudomonas solanacearum.

In general, the 6-substituted penicillins and the 7-substitutedcephalosporins provided by this invention demonstrate increased activityagainst the gram-negative microorganisms in comparison to theunsubstituted antibiotics. The activity vs the gram-positive organismson the other hand is somewhat less than that exhibited by theunsubstituted penicillins and cephalosporins. However, the especiallypreferred compounds provided herein, demonstrate increased activityagainst the gram-negative microorganisms while yet retaining theactivity against the gram-positive organisms which is demonstrated bythe unsubstituted cephalosporin antibiotics. The antibacterial activityof the especially preferred compounds is illustrated by the datapresented in Table I and II for two such preferred compounds.

The following Table I lists the minimum inhibitory concentrations (MIC)for two of the compounds of the invention against five clinical isolatesof penicillin resistant Staphylococcus aureus both in the presence andabsence of serum. The MIC values were determined by the Gradient Platemethod carried out essentially as described by Bryson and Szybalski,Science, 116, 45-46 (1952).

                                      Table I                                     __________________________________________________________________________    Antibiotic Activity vs. Penicillin Resistant                                  Staphylococcus aureus                                                                 Staphylococcus Clinical Isolates                                              MIC(mcg/ml)                                                                   V41    V32    X400   V84    X1.1                                      Compound.sup.1                                                                        NS.sup.2                                                                          S.sup.3                                                                          NS  S  NS  S  NS  S  NS  S                                     __________________________________________________________________________    A       4.2 6.7                                                                              4.5 7.6                                                                              16.6                                                                              15.4                                                                             3.1 2.0                                                                              0.5 <0.1                                  B       1.5 1.1                                                                              1.8 2.0                                                                              10.0                                                                               9.5                                                                             0.6 1.0                                                                              0.4 0.5                                   __________________________________________________________________________     .sup.1 A =                                                                    7-Mandelamido-7-methoxy-3-(1-methyl-1H-tetrazol-5-ylthiomethyl)-3-cephem-    -carboxylic acid.                                                              B =                                                                           7-[2-(2-Thienyl)acetamido]-7-methoxy-3-(1-methyl-1H-tetrazol-5-ylthiometh    l)-3-cephem-4-carboxylic acid.                                                 .sup.2 NS = absence of serum.                                                 .sup.3 S = presence of serum.                                            

In Table II which follows, the antibiotic activity against exemplarygram negative organisms is illustrated by the activity demonstrated bytwo of the preferred compounds. The activity, expressed in terms of theminimum inhibitory concentration, was determined by the Gradient Platetechnique.

                  Table II                                                        ______________________________________                                        Antibiotic Activity vs. Gram-Negative Microorganisms                                           Compound.sup.1                                                                MIC(mcg/ml)                                                  Organism           A          B                                               ______________________________________                                        Shigella sp.       2.0        6.6                                             Escherichia coli   2.5        7.9                                             Klebsiella pneumoniae                                                                            0.6        0.5                                             Aerobacter aerogenes                                                                             0.7        3.5                                             Salmonella heidelberg                                                                            0.6        0.9                                             Pseudomonas aeruginosa                                                                           >200       >200                                            Serratia marcescens                                                                              3.4        5.4                                             ______________________________________                                         .sup.1 A =                                                                    7-Mandelamido-7-methoxy-3-(1-methyl-1H-tetrazol-5-ylthiomethyl)-3-cephem-    -carboxylic acid.                                                              B =                                                                           7-[2-(2-Thienyl)acetamido]-3-(1-methyl-1H-tetrazol-5-ylthiomethyl)-3-ceph    m-4-carboxylic acid.                                                      

In the following Table III the antibiotic activity of the compounds ofthe invention is further illustrated by the activity shown forrepresentative compounds as determined by the standard disc-platemethod.

                  Table III                                                       ______________________________________                                        In vitro Antibiotic Spectrum Disc-Plate Method                                                Diameter of                                                                   Zone of Inhibition(mm)                                                        concentration(mg/ml)                                                          Test Compound.sup.1                                           Test Organism     C        D        E                                         ______________________________________                                        Staphylococcus aureus                                                                           24/0.5   24/0.5   25/0.1                                    Bacillus subtilis 25/0.5   24/0.5   22/0.1                                    Sarcina lutea     23/0.5   23/0.5   24/0.1                                    Mycobacterium avium                                                                             10/0.5   20/5.0   15/1.0                                    Proteus vulgaris  21/0.5   18/0.5   18/0.1                                    Salmonella gallinarum                                                                           26/0.5   25/0.5   20/1.0                                    Escherichia coli  23/0.5   20/0.5   22/0.1                                    Klebsiella pneumoniae                                                                           19/0.5   17/0.5   16/0.1                                    Serratia marcescens                                                                             19/0.5   17/0.5   18/1.0                                    Pseudomonas solanacearum                                                                        16/0.5   23/0.5   16/1.0                                    ______________________________________                                         .sup.1 C =                                                                    7-[2-(1-tetrazolyl)acetamido]-7-methoxy-3-(5-methyl-1,3,4-thiadiazol-2-yl    hiomethyl)-3-cephem-4-carboxylic acid                                          D =                                                                           7-[2-(1-Tetrazolyl)acetamido]-7-methoxy-3-acetoxymethyl-3-cephem-4-carbox    lic acid.                                                                      E =                                                                           7-Mandelamido-7-methoxy-3-(5-methyl-1,3,4-thiadiazol-2-ylthiomethyl)-3-ce    hem-4-carboxylic acid.                                                    

The substituted penicillins and cephalosporins provided by thisinvention are useful antibiotic compounds which are effective incombatting infections in warm blooded mammals. When administeredparenterally at a non-toxic dose between about 2.5 and 750 mg./kg. ofbody weight, the compounds of the invention are effective in combattingbacterial infections in warm-blooded mammals. As is recognized withother antibiotics, the dose required in the treatment of a particularhost varies. For example, depending on such circumstances as theseverity of the infection, the general health and physical condition ofthe particular host and the variability in response of the particularhost, the amount of antibiotic administered will vary. Likewise, atreatment regime comprising multiple doses, for example, 3 or 4 dosesadministered daily, may be desirable with a particular host.Alternatively, a compound of the invention can be administered as asingle daily dose, which regime may continue until the desired responsehas been attained.

The compounds of the invention, represented by the Formula I, which areesters other than the acetoxymethyl esters, or which contain a protectedhydroxy or a protected amino function do not possess antibiotic activityto any appreciable degree. However, by removal of the ester group or byremoval of the hydroxy and amino function protecting groups, byemploying well known methods and procedures, the antibiotic compounds ofthe invention are obtained where in the Formula I R₁ is hydrogen and afree amino or free hydroxy group is present.

The following examples more fully illustrate the present inventionwithout any intention to be limiting thereof.

In the following examples, infared absorption spectrum and nuclearmagnetic resonance spectrum are abbreviated IR and NMR respectively.Only the significant IR absorption attributable on the carbonyl functionof the β-lactam ring is given. Likewise, the pertinent peaks observed inthe NMR spectra are listed. The nuclear magnetic resonance spectra wereobtained on a Varian Associates T-60 Spectrometer with tetramethylsilaneas the reference standard. The values listed are in cycles per second(c.p.s.).

The following standard abbreviations are employed for the observed peaksin the NMR spectra: s = singlet; m = multiplet; q = quartet; d =doublet.

EXAMPLE 1

To 25 ml. of dry tetrahydrofuran maintained at ice bath temperature wereadded 2.2 ml. of methyl lithium (1.58 M.) and 4 ml. of dry methanol. Thesolution was stirred under nitrogen for about 2 minutes and was thencooled to a temperature of about -80° C. by means of a dry ice-acetonebath. To the cold solution was added a solution of 531 mg. ofp-nitrobenzyl 7-[2-(2-thienyl) acetamido]cephalosporanate in 8 ml. ofdry tetrahydrofuran and the reaction mixture stirred in the cold for 2minutes. To the cold reaction mixture was then added 0.143 ml. (1.2equivalents) of t-butyl hypochlorite, and the reaction mixture wasstirred for 10 minutes after the addition of the chlorinating agent wascomplete. The reaction mixture was then quenched by adding 4 ml. ofglacial acetic acid. The reaction mixture was then evaporated in vacuoand the residue dissolved in dichloromethane. The solution of theresidue was washed three times with a saturated solution of sodiumchloride, one time with a dilute solution of sodium thiosulfate, twotimes with a solution of sodium bicarbonate and thereafter one time witha saturated solution of sodium chloride. The washed solution was thendried and evaporated in vacuo to provide 536 mg. of substantially purep-nitrobenzyl 7-methoxy-7-[2-(2-thienyl)acetamido]cephalosporanate.

I.r. (kbr) 1780^(cm-1)

N.m.r. (cdcl₃):

200-210 (m, 2H, C₂),

298 (q, 2H, --CH₂ OAc),

3 302 (s, 1H, C₆)

231 (s, 2H, amide methylene)

4.3-436 (m, 3H, thienyl protons)

420 (s, 2H, ester methylene)

470 (q, 4H, aromatic)

123 (s, 3H, acetoxy methyl)

207 (m, 3H, 7-methoxy).

The p-nitrobenzyl ester was cleaved to provide the free acid in thefollowing manner. A suspension of 260 mg. of 5 percent Pd/C in 15 ml. of1:1 methanol:tetrahydrofuran was prehydrogenated for one hour. To thesuspension was added a solution of 260 mg. of the p-nitrobenzyl ester in15 ml. of 1:1 methanol:tetrahydrofuran and the mixture was hydrogenatedfor 3 hours in an atmosphere of hydrogen gas. The catalyst was filteredand the filtrate was evaporated to provide a solid residue. The residuewas dissolved in ethyl acetate and the acidic reduction product wasextracted with a dilute solution of sodium bicarbonate. The bicarbonateextracts were made acid to pH 2.5 by the addition of dilute hydrochloricacid and were then extracted with ethyl acetate. The extact was driedand thereafter evaporated in vacuo to yield 182 mg. of7-methoxy-7-[2-(2-thienyl)acetamido]cephalosporanic acid.

I.r. (kbr): 1778^(cm-1)

Nmr (100 mcg.):

285-296 (m, 2H, C₂ ),

496-515 (m, 2H, --CH₂ --OAc),

496-515 (m, IH, C₆),

693-705 (m, IH, amide NH),

391 (s, 2h, amide methylene),

696-728 (m, 3H, thienyl protons),

158 (s, 3H, acetoxy methyl)

348 (m, 3H, 7-methoxy).

EXAMPLE 2

To 25 ml. of cold, dry, tetrahydrofuran maintained under an atmosphereof nitrogen were added 2.2 ml. of 1.58 M. methyl lithium and 4 ml. ofdry methanol. The solution was stirred for several minutes and was thencooled to about -80° C. by means of a dry ice-acetone bath. To the coldsolution was added 480 mg. of benzhydryl 7-acetamidocephalosporanate in8 ml. of dry tetrahydrofuran. The reaction mixture was stirred forapproximately 2 minutes and then 0.143 ml. (1.2 equivalents) of t-butylhypochlorite was added with stirring. After the reaction mixture wasstirred for approximately 10 minutes, the reaction was quenched by theaddition of 4 ml. of glacial acetic acid. The reaction mixture wasevaporated to dryness and the residue was dissolved in dichloromethane.The solution was washed successively with a saturated solution of sodiumchloride, a dilute solution of sodium thiosulfate, a saturated solutionof sodium bicarbonate, and a saturated solution of sodium chloride. Thewashed solution was then dried and evaporated to dryness to yield 541mg. of crude benzhydryl 7-acetamido-7-methoxycephalosporanate.

I.r. (chcl₃): 1790^(cm-1)

N.m.r. (acetone d₆):

304 (s, IH, C₆)

209 (s, 3H, 7-methoxy)

The benzhydryl ester was cleaved by reacting the ester with a 50:50mixture of trifluoroacetic acid and 98% formic acid to provide a solidresidue containing 7-acetamido-7-methoxycephalosporanic acid.

N.m.r. (acetone d₆):

306 (s, IH, C₆)

209 (s, 3H, 7-methoxyl).

EXAMPLE 3

In accordance with the procedures described by Example 1, benzhydryl3-methoxymethyl-7-[2-(2-thienyl)-acetamido]-3-cephem-4-carboxylate wasreacted at a temperature of -80° C. with methyl lithium in the presenceof methanol and t-butyl hypochlorite to provide benzhydryl3-methoxymethyl-7-methoxy-7-[2-(2-thienyl)acetamido]-3-cephem-4-carboxylate.

I.r. (kbr): 1780^(cm-1)

N.m.r. (cdcl₃):

303 (s, IH, C₆)

200-210 (m, 3H, 7-methoxyl).

The above ester was hydrolyzed under acidic conditions in the followingmanner. To 150 mg. of the ester in a 50 ml. round bottom flaskmaintained at a temperature of approximately -10° C. was added 2.5 ml.of a 1:1 mixture of trifluoroacetic acid:formic acid. To the stirredmixture was added 30 ml. of dichloromethane. The reaction mixture wasthen evaporated to remove solvents and the residue was dissolved inethyl acetate. The antibiotic acid was extracted from the ethyl acetatesolution with a saturated solution of sodium bicarbonate. Thebicarbonate extract was acidified to pH 2.5 by the addition ofhydrochloric acid and the acidified solution was thereafter extractedwith ethyl acetate. The ethyl acetate extract was dried and thenevaporated in vacuo to provide 60 mg. of3-methoxymethyl-7-methoxy-7-[2-(2-thienyl)-acetamido]-3-cephem-4-carboxylicacid.

N.m.r. (cdcl₃):

304 (s, IH, C₆)

200-210 (m, 3H, 7-methoxyl).

EXAMPLE 4

To 25 ml. of dry tetrahydrofuran maintained at about 0° C. was added 3ml. of 1.58 M. methyl lithium and 6 ml. of dry methanol. The solutionwas stirred for about 5 minutes and was then cooled to approximately-80° C. by means of a dry ice-acetone bath. To the cold solution wasadded a solution of 582 mg. of p-nitrobenzyl3-methyl-7-(2-t-butyloxycarboxamido-2-phenylacetamido)-3-cephem-4-carboxylatein 8 ml. of dry tetrahydrofuran and the reaction mixture stirred for 2minutes following the addition. To the reaction mixture was then added0.143 ml. of t-butyl hypochlorite and the reaction mixture was stirredfor 10 minutes following the addition of the hypochlorite. The reactionwas then quenched by the addition of 6 ml. of 98% formic acid. Thequenched reaction mixture was evaporated in vacuo and the residuedissolved in dichloromethane. The solution was washed consecutively witha saturated solution of sodium chloride, dilute aqueous sodiumthiosulfate, a saturated solution of sodium bicarbonate and finally witha saturated sodium chloride solution. The washed solution was thenevaporated in vacuo to dryness to provide p-nitrobenzyl7-methoxy-3-methyl-7-(t-butyloxycarboxamido-2-phenylacetamido)-3-cephem-4-carboxylate.

I.r. (kbr): 1785 cm⁻¹

N.m.r. (cdcl₃):

212 (s, 3H, 7-methoxyl)

304 (s, IH, C₆)

To a solution of 350 mg. of the amino protected ester prepared asdescribed above in a 1:1 mixture of methanol: tetrahydrofuran was added350 mg. of 5% palladium or carbon catalyst which had been prereduced.The suspension was stirred in an atmosphere of hydrogen for 3 hours atroom temperature. The catalyst was filtered, and the filtrate evaporatedto dryness. The residue obtained was dissolved in ethyl acetate and thesolution was washed with an aqueous solution of sodium bicarbonate. Thebicarbonate wash was acidified to pH 2.5 with hydrochloric acid and theacidified solution was extracted with ethyl acetate. The ethyl acetateextract was dried over sodium sulfate and thereafter evaporated todryness to yield 210 mg. of3-methyl-7-methoxy-7-(2-t-butyloxycarbamido-2-phenylacetamido)-3-cephem-4carboxylicacid. The acid thus obtained was dissolved in 2 ml. of coldtrifluoroacetic acid and the solution stirred for 5 minutes. The coldacid solution was then added by dropwise addition to diethyl ether toprecipitate the trifluoroacetic acid salt of3-methyl-7-methoxy-7-(2-amino-2-phenylacetamido)-3-cephem-4-carboxylicacid.

N.m.r. (100 mcg., D₂ O);

394 (s, 3H, 7-methoxyl)

561 (s, IH, C₆).

EXAMPLE 5

According to the procedure described by Example 4, 479 mg. of2,2,2-trichloroethyl 3-methyl-7-phenoxyacetamido-3-cephem-4-carboxylatewas reacted with methanol, methyl lithium and t-butyl hypochlorite inanhydrous tetrahydrofuran at -80° C. to provide 2,2,2-trichloroethyl3-methyl-7-methoxy-7-phenoxy-acetamido-3-cephem-4-carboxylate.

I.r. (chcl₃): 1780 cm⁻¹

N.m.r. (cdcl₃): 214 (s, 3H, 7-methoxyl)

The trichloroethyl ester prepared as described above was hydrolyzed inthe following manner. To a cold, 0° C. solution of 200 mg. of the esterin 1 ml. of dimethylformamide was added 0.3 ml. of 98% formic acid and220 mg. (9 equivalents) of zinc dust, and the mixture stirred for 1.5hours. The zinc was filtered and washed with water and ethyl acetate.The water layer of the filtrate was acidified to pH 2.5 by the additionof 5% hydrochloric acid. The ethyl acetate layer and the acidifiedaqueous layer were shaken vigorously and separated. The ethyl acetatelayer was then washed with aqueous sodium bicarbonate and thebicarbonate wash was then acidified to pH 2.5 by the addition ofhydrochloric acid. The acidified was was extracted with ethyl acetateand the extract dried and then evapoated in vacuo to give 70 mg. of3-methyl-7-methoxy-7-phenoxyacetamido-3-cephem-4-carboxylic acid.

N.m.r. (cdcl₃):

213 (s, 3H, 7-methoxyl)

305 (s, IH, C₆).

EXAMPLE 6

To 200 ml. of dry tetrahydrofuran maintained at 0 to 5° C. was addedwith stirring 20 ml. of 1.58 M. methyl lithium. To this solution wasadded cautiously 40 ml. of dry methanol with stirring. The solution wasthen cooled with stirring to -80° C. by means of a dry ice-acetone bath.To the cold solution was added by dropwise addition under nitrogen asolution of 4.69 g. of p-nitrobenzyl 6-phenylacetamidopenicillanate in80 ml. of dry tetrahydrofuran. The reaction mixture was stirred for 3minutes in the cold and then 1.43 ml. (1.2 equivalents) of t-butylhypochlorite was added with stirring. The reaction was allowed to stirfor about 25 minutes and was then quenched by the addition of 40 ml. of98% formic acid.

The reaction mixture was then evaporated in vacuo to a volume of 50 ml.The liquid concentrate was poured into a saturated solution of sodiumchloride which was layered with dichloromethane. The organic layer wasseparated and the aqueous salt layer was washed with dichloromethane.Both dichloromethane layer were combined and washed with a saturatedsolution of sodium chloride, a saturated solution of sodium bicarbonate,and finally again with a saturated sodium chloride solution. The washedorganic layer containing the reaction product mixture was then driedover sodium sulfate and evaporated to provide 5.1 g. of crude reactionproduct mixture.

The crude reaction product mixture, 5.1 g., was dissolved in 100 ml. ofbenzene and the solution was chromatographed over a column packed with300 g. of silica/15% water. The column was eluted initially with 10%benzene in ethyl acetate. The concentration of benzene was graduallyincreased to 20% benzene. Sixty fractions of 175 ml. volume werecollected. Fractions 34 to 45 and 47 to 50 were combined and evaporatedto dryness to provide 2.83 g. of p-nitrobenzyl6-methoxy-6-phenylacetamidophenicillanate.

I.r. (chcl₃): 1778 cm³¹ 1

N.m.r. (cdcl₃):

84 (s, 6H, 2,2-dimethyl)

267 (s, IH, C₃)

333 (s, IH, C₅)

396 (s, IH, amide proton)

219 (s, 2H, amide methylene)

438 (s, 5H, phenyl protons)

316 (s, 2H, ester methylene)

468 (q, 4H, nitrophenyl protons)

204 (s, 3H, 6-methoxyl)

A solution of 200 mg. of the above ester in 15 ml. of 1:1tetrahydrofuran:methanol was added to a suspension of 200 mg. of 5%palladium on carbon in a 1:1 mixture of tetrahydrofuran:methanol whichhad been prehydrogenated for 1 hour. The solution was agitated in anatmosphere of hydrogen at room temperature for 3 hours. The catalyst wasfiltered and the filtrate evaporated in vacuo to obtain a solid residue.The residue was dissolved in ethyl acetate and the solution wasextracted with a saturated solution of sodium bicarbonate. Thebicarbonate extract was separated and acidified to pH 2.0 with dilutehydrochloric acid. The acidified solution was then extracted with ethylacetate and the extract dried over sodium sulfate. The dried extract wasthen evaporated to dryness to yield 130 mg. of6-methoxy-6-phenylacetamidopenicillanic acid.

N.r.r. (cdcl₃):

85 (d, 6H, 2,2-dimethyl)

261 (s, IR, C₃)

332 (s, IH, C₅)

615 (s, IH, amide proton)

EXAMPLE 7

According to the alkoxylation procedure described by the foregoingexamples, 584 mg. of p-nitrobenzyl6-(2-t-butyloxycarbamido-2-phenylacetamido)penicillanate was reactedwith methyl lithium, methanol, and t-butyl hypochlorite in anhydroustetrahydrofuran at -80° C. to provide following the workup employed inthe foregoing examples, 512 mg. of p-nitrobenzyl6-methoxy-6-(2-t-butyloxycarbamido-2-phenylacetamido)penicillanate.

N.m.r. (cdcl₃):

211 (s, 3H, 6-methoxy)

337 (s, IH, C₅).

To a solution of 205 mg. of the methoxylated ester obtained as describedabove in 15 ml. of a 1:1 mixture of tetrahydrofuran and methanol wasadded a suspension of 205 mg. of 5% palladium on carbon catalyst in a1:1 mixture of tetrahydrofuran and methanol which ws previouslyhydrogenated. The suspension was reacted with hydrogen at roomtemperature for 3hours. The catalyst was filtered and the filtrate wasevaporated in vacuo. The solid residue obtained was dissolved in ethylacetate and the acidic reaction product extracted therefrom with asaturated solution of sodium bicarbonate. The bicarbonate extract wasacidified to pH 2.5 with hydrochloric acid, and the acidic reactionproduct back extracted into ethyl acetate. The ethyl acetate extract wasdried and then evaporated in vacuo to yield 130 mg. of 6-methoxy6-(2-t-butyloxycarbamido-2-phenylacetamido)penicillanic acid.

To a solution of 110 mg. of the above prepared amino protectedpenicillanic acid in 25 ml. of acetonitrile was added a solution of 50mg. of p-toluene sulfonic acid in 5 ml. of acetonitrile. The reactionmixture was stirred at room temperature for 3 hours and was thereafterdiluted with 3 ml. of water. The pH of the solution was adjusted to pH4.7 with triethylamine and the volume of the reaction mixture wasreduced by one half. On standing in the refrigerator a crystallineprecipitate of 6-methoxy-6-(2-amino-2-phenylacetamido)penicillanic acidwas obtained.

EXAMPLE 8

According to the procedures described by the foregoing examples, 515 mg.of p-nitrobenzyl 6-(2,6-dimethoxybenzamido) penicillanate was reactedwith methyl lithium, methanol, and t-butyl hypochlorite in anhydroustetrahydrofuran at -80° C. to provide6-methoxy-6-(2,6-dimethoxybenzamido)penicillanic acid p-nitrobenzylester.

I.r. (chcl₃): 1778^(cm-1)

N.m.r. (cdcl₃):

216 (s, 3H, 6-methoxyl)

336 (s, IH, C₅).

Hydrogenolysis of the ester thus prepared, according to the procedurepreviously described, provided 93 mg. of6-methoxy-(2,6-dimethoxybenzamido)penicillanic acid.

N.m.r. (cdcl₃):

214 (s, 3H, 6-methoxyl)

332 (s, IH, C₅)

EXAMPLE 9

To 70 ml. of dry benzene containing 0.1 g. of p-toluene sulfonic acidwas added 1.14 g. of benzhydryl7-(2-hydroxy-2-phenylacetamido)cephalosporanate with stirring. To thestirred solution 0.144 g. of ethyl vinyl ether was slowly added bydropwise addition. The reaction mixture was evaporated in vacuo and theresidue dissolved in ethyl acetate. The solution was washed with coldwater, dried and evaporated to provide in a nearly quantitative yieldthe hydroxy protected cephalosporanic ester, benzhydryl7-[2-(1-ethoxyethoxy)-2-phenylacetamido]cephalosporanate.

The protected hydroxy cephalosporanic ester thus prepared was reactedaccording to the procedures described in the foregoing examples withlithium methoxide, methanol, and t-butyl hypochlorite in anhydroustetrahydrofuran at -80° C. to provide benzhydryl7-methoxy-7-[2-(1-ethoxyethoxy)-2-phenylacetamido]cephalosporanate. Theproduct was purified by chromatography over silica gel during whichpartial deblocking of the protected hydroxyl group occurred to provide amixture containing 35% of the purified reaction product and 20% of thede-blocked reaction product7-methoxy-7-(2-hydroxy-2-phenylacetamido)cephalosporanate. The purifiedmixture was reacted with a mixture of trifluoroacetic acid and 98%formic acid to effect removal of both the hydroxyl protecting group andthe benzhydryl ester group to provide7-methoxy-7-(2-hydroxy-2-phenylacetamido)cephalosporanic acid.

N.m.r. (cdcl₃)

122 (s, 3H, acetoxy methyl)

204 (s, 3H, 7-methoxyl).

EXAMPLE 10

According to the reaction procedures described by the foregoing Examplesthe following compounds are prepared with the indicated reactants. Ineach instance tetrahydrofuran is the preferred solvent and t-butylhypochlorite is the preferred chlorinating agent. Listed in the order ofappearance are the reaction product, the alkali metal alkoxide, thecorresponding alcohol and the starting penicillin ester of cephalosporinester.

3-methylthiomethyl-7-ethoxy-7-(2-amino-2-phenylacemido)-3-cephem-4-carboxylicacid; lithium ethoxide; absolute ethanol;p-nitrobenzyl3-methylthiomethyl-7-(2-amino-2-phenylacetamido)-3-cephem-4-carboxylate.

3-carbamoyloxymethyl-7-isopropoxy-7-(2-phenoxyacetammido)-3-cephem-4-carboxylicacid; lithium isopropoxide; isopropyl alcohol; 2,2,2-trichloroethyl3-carbamoyloxmethyl-7-(2-phenoxy acetamido)-3-cephem-4-carboxylate.

7-Allyloxy-3-methyl-7-[2-(2-thienyl)acetamido]-3-cephem--4-carboxylicacid; potassium allyloxide; allyl alcohol; benzhydryl3-methyl-7-[2-(2-thienyl)acetamido]-3-cephem-4-carboxylate.

7-cyclopropoxy-3-ethoxymethyl-7-(2-phenoxyacetamido)-3-cephem-4-carboxylate;lithium cyclopropoxie; cyclopropyl alcohol; p-nitrobenzyl3-ethoxymethyl-7-(2-phenoxyacetamido)-3-cephem-4-carboxylate.

7-benzyloxy-3N-methylcarbamoyloxymethyl-7-(2-phenyl-mercaptoacetamido)-3-cephem-4-carboxylicacid; lithium benzyloxide; benzyl alcohol; 2,2,2-trichloroethyl3-N-methylcarbamoyloxymethyl-7-(2-phenylacetamido)-3-cephem-4-carboxylate.

EXAMPLE 11

To 25 ml. of anhydrous tetrahydrofuran maintained in an atmosphere ofnitrogen and at a temperature of 0° C. were added with stirring 2.2 ml.of 1.65 N methyl lithium and 4 ml. of dry methanol. The solution wasstirred for 5 minutes and was then cooled to a temperature of -80° C. Tothe cold solution was added 573 mg. of benzhydryl7-(2-hydroxy-2-phenylacetamido)-3-carbamoyloxymethyl-3-cephem-4-carboxylateand stirring was continued for several minutes before 0.143 ml. oft-butyl hypochlorite was added. The reaction mixture was stirred for anadditional 20 minutes at -80° C. before the reaction was quenched by theaddition of 4 ml. of glacial acetic acid. The reaction mixture was thenevaporated to remove solvents and the residue dissolved indichloromethane. The solution of the residue was washed consecutivelywith a saturated solution of sodium chloride, a dilute solution ofsodium thiosulfate, a saturated solution of sodium bicarbonate andfinally again with a saturated solution of sodium chloride. The washedsolution was dried and evaporated to provide 604 mg. of the crudereaction product mixture containing benzhydryl3-carbamoyloxymethyl-4'-oxo-5'-phenylspiro-[3-cephem-7,2'-oxazolidine]-4-carboxylate.The crude mixture was purified on silica coated preparative thin layerplates using a 50:50 ethyl acetate:benzene solvent system fordevelopment. The spot on the chromatogram containing the reactiveproduct was scraped from the plate and the product was separated fromthe silica by extraction with ethyl acetate to provide 375 mg. of thepurified benyhydryl ester.

EXAMPLE 12

To 1 ml. of a cold 50:50 mixture of trifluoroacetic acid and 98% formicacid was added 100 mg. of the purified benzhydryl ester prepared asdescribed by Example 11. After one minute, 10 ml. of dichloromethanewere added and the solution was evaporated under vacuum to a smallvolume. The concentrate was dissolved in ethyl acetate and the acidicreaction product was extracted with an aqueous solution of sodiumbicarbonate. The bicarbonate extract was acidified to pH 2.5 and thereaction product was back extracted with ethyl acetate. The extract wasdried and evaporated to obtain 66 mg. of3-carbamoyloxymethyl-4'-oxo-5'-phenylspiro-[3-cephem-7,2'-oxazolidine]-4-carboxylicacid as a solid in substantially pure form.

EXAMPLE 13

To a solution of 1.5 g. of sodium7-methoxy-7-(2-phenoxyacetamido)-3-acetoxymethyl-3-cephem-4-carboxylatein 50 ml. of water was added 1 equivalent of sodium hydroxide and 1equivalent of 2-thiol-1,3,4-thiadiazole. The mixture was heated at atemperature of 70° C. for five hours and was then acidified with 1Nhydrochloric acid. The reaction product was extracted from the acidifiedmixture with ethyl acetate and the extract was washed with water andevaporated to dryness. The solid residual product was crystallized froma mixture of ethyl acetate and DMF to yield7-methoxy-7-(2-phenoxyacetamido)-3-(1,3,4-thiadiazol-2-ylthiomethyl)-3-cephem-4-carboxylicacid in substantially pure form.

EXAMPLE 14

According to the procedure described by Example 13, sodium7-ethoxy-7-[2-(2-thienyl)acetamido]-3-acetoxymetyl-3-cephem-4-carboxylatewas reacted in water with 2-thiol-1-methyl-1H-tetrazole to yield7-[2-(2-thienyl)acetamido]-3-(1-methyl-1H-tetrazol-5-ylthiomethyl)-3-cephem-4-carboxylicacid.

EXAMPLE 157-Methoxy-7-mandelamido-3-(5-methyl-1,3,4-thiadiazol-2-ylthiomethyl-3-cephem-4-carboxylicacid

To a solution of 912 mg of sodium7-methoxy-7-mandelamido-3-acetoxymethyl-3-cephem-4-carboxylate, preparedby the method described by Example 9, in 1 ml of water was added asolution of sodium hydroxide to adjust the pH of the solution to pH 6.5.To the solution was added 10 mg of 2-mercapto-5-methyl-1,3,4-thiadiazoleand the reaction mixture was heated with stirring for 3 hours at 70° C.The reaction mixture was cooled and was then acidified with dilutehydrochloric acid. The acidified mixture was extracted with ethylacetate containing 10% by volume of tetrahydrofuran and was then driedover magnesium sulfate and evaporated to dryness to yield the reactionproduct mixture containing7-methoxy-7-mandelamido-3-(5-methyl-1,3,4-thiadiazol-2-ylthiomethyl)-3-cephem-4-carboxylicacid.

The dry residue was dissolved in tetrahydrofuran and 400 mg ofdiphenyldiazomethane were added to the solution. The solution wasstirred at room temperature for 12 hours and was then evaporated to asolid residue. The residue was leeched with a mixture ofchloroform:petroleum ether which on evaporation to dryness afforded onegram of a solid foam residue.

The residue was chromatographed on preparative thin layer silica gelplates with ethyl acetate:benzene (50:50), to yield 229 mg of thediphenylmethyl ester of the starting material, and 188 mg ofdiphenylmethyl7-methoxy-7-mandelamido-3-(5-methyl-1,3,4-thiadiazol-2-ylthiomethyl)-3-cephem-4-carboxylate.

The diphenylmethyl ester of the reaction product, 188 mg, was dissolvedin 2 ml of 97 percent formic acid and the solution was stirred at roomtemperature for 75 minutes. The reaction mixture was treated withbenzene and evaporated. More benzene was added to the residue and themixture was again evaporated to dryness. The dry residue was partiallydissolved in ethyl acetate and a saturated solution of sodiumbicarbonate was added. The bicarbonate layer was separated, washed withethyl acetate and acidified to pH 2.0 with hydrochloric acid. Theacidified solution was then extracted with ethyl acetate containing 10%by volume of tetrahydrofuran. The extract was dried and was evaporatedto dryness to yield 76 mg of7-methoxy-7-mandelamido-3-(5-methyl-1,3,4-thiadiazol-2-ylthiomethyl)-3-cephem-4-carboxylicacid having the following NMR spectrum (acetone d₆):

215 (q, 2H, C₂)

267 (q, 2H, C₃)

306 (s, 1H, C₆)

205 (s, 3H, C₇ methoxy)

492 (s, 1H, amide N-H)

315 (s, 1H, amide C-H)

375 (s, 2H, C₃ CH₂ -S)

430-460 (m, 5H, aromatic)

162 (s, 3H, thiadiazole-5-methyl group).

EXAMPLE 167-Methoxy-7-[2-(2-thienyl)acetamido]-3-(1-methyltetrazol-5-ylthiomethyl)-3-cephem-4-carboxylicacid

To a solution of 900 mg of sodium7-methoxy-7-[2-(2-thienyl)acetamido]-3-acetoxymethyl-3-cephem-4-carboxylate,prepared by the method described in Example 1, in 10 ml of water wasadded a solution of sodium hydroxide to adjust the pH of the solution topH 6.5. To the solution was added 232 mg of 1-methyltetrazole-5-thioland the mixture was heated with stirring for 3 hours at 70° C.

The pH of the reaction mixture was adjusted to pH 7.0 and was thenwashed with ethyl acetate. The pH of the reaction mixture was nextadjusted to pH 2.5 with dilute hydrochloric acid and was then extractedwith ethyl acetate containing 20% by volume of THF. The extract wasdried over magnesium sulfate and was then evaporated to dryness to yield436 mg of solid reaction product mixture.

The residue of reaction product was dissolved in 15 ml of THF and to thesolution was added a solution of 400 mg of diphenyldiazomethane in 5 mlof THF. The esterification mixture was stirred for 12 hours at roomtemperature and was then evaporated to afford 650 mg of the crudeesterified reaction product mixture.

The esterified reaction product mixture was chromatographed overpreparative thin layer silica gel plates with 30% ethyl acetatebenzeneto afford 116 mg of the diphenylmethyl ester of the starting materialand 104 mg of the reaction product, diphenylmethyl 7-methoxy7-[2-(2-thienyl)/acetamido]-3-(1-methyltetrazol-5-ylthiomethyl-3-cephem-4-carboxylate.

The 104 mg of esterified reaction product were dissolved in 1 ml of acold (0° C.) mixture of equal volumes of THF and 97% formic acid and themixture was allowed to warm to room temperature. The reaction mixturewas then treated with 75 ml of methylene chloride and the solution wasevaporated to dryness. The residue was dissolved in 20 ml of ethylacetate and the solution was extracted three times with 20 ml portionsof a saturated solution of sodium bicarbonate. The bicarbonate extractswere combined and were acidified to pH 2.5. The reaction product wasextracted from the acidified bicarbonate extracts with 10% ethanol/ethylacetate and the extract was dried and evaporated to yield 66 mg of thereaction product,7-methoxy-7-[2-(2-thienyl)acetamido]-3-(1-methyltetrazol-5-ylthiomethyl)-4-carboxylicacid having the following NMR spectrum (acetone d₆).

220 (s, 2H, C₂)

266 (s, 2H, C₃)

305 (s, 1H, C₆)

209 (s, 3H, C₇ methoxy)

234-246 (m, 5H, amide CH₂, tetrazole methyl H)

421-460 (m, 5H, carboxy H, thienyl H and N-H)

I.r. (chcl₃) 1782^(cn-1) β-lactam carbonyl

EXAMPLE 177-Methoxy-7-mandelamido-3-(1-methyltetrazol-5-ylthiomethyl)-3-cephem-4-carboxylicacid

Sodium 7-methoxy-7-mandelamido-3-acetoxymethyl-3-cephem-4-carboxylate,1.3 mmole, prepared as described in Example 9, was dissolved in 15 ml.of water and the pH of the solution was adjusted to pH 6.5 by theaddition of 0.1 N sodium hydroxide. To the solution was then added 161mg. of 1-methyltetrazole-5-thiol and the solution was heated withstirring for 3 hours at 70° C. The reaction mixture was cooled to roomtemperature and to the mixture was added 50 ml of 20% THF/ethyl acetate.The pH of the mixture was adjusted to pH 2.0 with dilute hydrochloricacid and the organic layer was separated. The aqueous layer was washedtwice with 20% THF/EtOAc and the organic layers were combined. Theorganic layers were dried over sodium sulfate and were evaporated toyield 570 mg of crude reaction product mixture.

The crude product was dissolved in 25 ml of THF and 400 mg ofdiphenyldiazomethane were added to the solution. The esterificationmixture was stirred at room temperature for two hours and was thenevaporated to dryness. The residue was leeched with achloroform-petroleum ether mixture. The solvents were evaporated toyield 703 mg of esterified reaction product mixture.

The esterified mixture was chromatographed on preparative thin layersilica gel plates using ethyl acetate:benzene (50:50) to provide 120 mgof diphenylmethyl7-methoxy-7-mandelamido-3-(1-methyltetrazol-5-ylthiomethyl)-3-cephem-4-carboxylate.

The ester, 100 mg. was dissolved in 1 ml of a 50:50 mixture of THF and97% formic acid at 0° C. The mixture was allowed to warm to roomtemperature and was then evaporated to dryness. Ethyl acetate was addedto dissolve the residue and the solution was extracted twice with asaturated solution of sodium bicarbonate. The bicarbonate extracts werecombined and were then acidified with dilute hydrochloric acid to pH2.5. The product was extracted from the acidified bicarbonate extractswith ethyl acetate. The ethyl acetate was dried and evaporated to yield53 mg of product,7-methoxy-7-mandelamido-3-(1-methyltetrazole-5-ylthiomethyl)-3-cephem-4-carboxylicacid having the following NMR spectrum (60 megacycles run in acetone d₆)

218 (g, 2H, C₂)

265 (g, 2H, C₃ methylene)

305 (s, 1H, C₆)

205 (s, 3H, C₇ methoxy)

432-455 (m, 6H, phenyl and N-H)

310-317 (m, 2H, OH, and amide methine)

240 (s, 3H, tetrazole methyl).

EXAMPLE 18

To a 25 ml of tetrahydrofuran maintained at 0° C. was added 2 ml of a1.81 molar solution of methyl lithium in ether and 4 ml of methanol. Thesolution was cooled to -80° C. in a dry ice-acetone bath and 700 mg ofdiphenylmethyl7-[2-(1-ethoxyethoxy)-2-phenylacetamido]-3-(1-methyltetrazol-2-ylthiomethyl)-3-cephem-4-carboxylatewere added. Next, .155 ml of t-butyl hypochlorite were added and thereaction mixture was stirred for 15 minutes. Glacial acetic acid, 4 ml,was added and the reaction mixture was evaporated to dryness. Thereaction product was isolated from the residual reaction product mixtureaccording to the work-up procedures described in previous examples toprovide 209 mg of benzhydryl3-methoxy-7-[2-(1-ethoxyethoxy)-2-phenylacetamido]-3-(1-methyltetrazol-2-ylthiomethyl)-3-cephem-4-carboxylaterepresented by the following formula. ##STR22##

The reaction product was dissolved in 2 ml of a 50:50 mixture of TFA and97% formic acid at 0° C. The solution was allowed to warm to roomtemperature and was then evaporated to dryness. The residue wasdissolved in ethyl acetate and the solution was extracted with asaturated solution of sodium bicarbonate. The bicarbonate extract wasacidified to pH 2.5 with hydrochloric acid and the product was extractedwith ethyl acetate. The ethyl acetate extract was dried and wasevaporated to dryness to yield7-methoxy-7-mandelamido-3-(1-methyltetrazol-5-ylthiomethyl)-3-cephem-4-carboxylicacid having the same NMR and IR spectrum as the product obtained by themethod described in Example 17.

EXAMPLE 19

To 25 ml of tetrahydrofuran was added 2 ml of a 1.8 molar solution ofmethyl lithium in ether. The solution was cooled to 0° C. and 4 ml ofmethanol were added to the solution. The solution was then cooled to-80° C and 468 mg of acetoxymethyl7-[2-(2-thienyl)acetamido]-3-acetoxymethyl-3-cephem-4-carboxylate wereadded, followed by the addition of 0.143 ml of t-butylhypochlorite. Thecold reaction mixture was stirred for 25 minutes and was thereafterdiluted with 4 ml of glacial acetic acid. The acidified reaction mixturewas evaporated to dryness and the residual reaction product mixture wasdissolved in dichloromethane. The solution was washed consecutively witha saturated solution of sodium chloride, a dilute solution of sodiumthiosulfate, a saturated solution of sodium bicarbonate, and finallywith a saturated solution of sodium chloride. The washed solution wasdried and was then evaporated to dryness. The residue waschromatographed on preparative thin layer silica gel plates usingbenzene-ethyl acetate (7:3). The location of separated materials on thechromatogram was determined with an ultraviolet lamp. The reactionproduct was eluted from the silica gel with acetone. The acetone wasevaporated to yield 202 mg of acetoxymethyl7-methoxy-7-[2-(2-thienyl)acetamido]-3-acetoxymethyl-3-cephem-4-carboxylate.

I claim:
 1. The process for preparing a substituted β-lactam compound ofthe formula ##STR23## which comprises a) reacting in an inert, anhydroussolvent at a temperature between -90° and -15° C. a β-lactam ester ofthe formula ##STR24## with between 2 and 6 equivalents per 1 equivalentof β-lactam ester of an alkali metal salt of the formula

    M.sup.+- 0-R.sub.2.

in the presence of an excess of the corresponding alcohol of the formulaHOR.sub. 2 ; b) adding between 1 and 5 equivalents of a halogenatingagent; c) acidifying said mixture; and d) recovering the substitutedβ-lactam ester from the reaction mixture; where in the precedingformulae R is hydrogen, C₁ -C₆ alkyl, 4-amino-4-carboxybutyl, phenyl,substituted phenyl, or a group of the formula ##STR25## wherein P isphenyl, C₁ -C₄ lower alkylphenyl, halophenyl, hydroxyphenyl, C₁ -C₄lower alkoxyphenyl, 2-thienyl, 3-thienyl, 2-furyl, 3-furyl, or1-tetrazyl; Z is an oxygen atom, or a sulfur atom; n is 0 or 1; a ishydrogen or C₁ -C₃ lower alkyl, b is hydrogen, C₁ -C₃ lower alkyl,protected hydroxy, amino, or protected amino; and when n is 1, P isphenyl, C₁ -C₄ lower alkylphenyl, halophenyl, hydroxyphenyl, C₁ -C₄lower alkoxyphenyl and b is hydrogen or C₁ -C₃ lower alkyl; R₁ ishydrogen, an alkali metal cation or a readily removable ester formingmoiety; and R₂ is C₁ -C₄ lower alkyl, allyl, propargyl, C₃ -C₆cycloalkyl, or benzyl.
 2. The process of claim 1 wherein thechlorinating agent is t-butyl hypochlorite.
 3. The process of claim 1wherein R₂ is C₁ -C₄ lower alkoxy.